The plasma membrane (outer membrane) of intact eukaryotic cells is characterized by a highly organized structure. This high level of membrane organization is determined, among others, by the molecular structure of the is specific lipids constituting the membrane; the ratio between the various lipid species from which the membrane is composed; the distribution of the phospholipids between the outer and inner leaflets of the membrane; and by the membrane protein constituents.
While maintenance of the high level of membrane organization is fundamental to normal cell physiology, substantial perturbations and alterations of the normal organization of the cell plasma membrane (PNOM) occur in cells undergoing a death process, especially by apoptosis, and in platelets undergoing activation during blood coagulation. Such alterations and perturbations may be evident both at the morphological level (e.g., membrane blebbing) and at the molecular level. The scope of these perturbations occurring during cell death or platelet activation is not fully elucidated. They include, among others, scrambling and redistribution of the membrane phospholipids, with movement to the cell surface of aminophsopholipids, mainly phosphatidylserine (PS) and phosphatidylethanolamine (PE), which are normally restricted almost entirely to the inner leaflet of the membrane bilayer, and reciprocal movement of sphingomyelin (SM) and phosphatidylcholine (PC) from the outer leaflet to the inner leaflet of the membrane. PNOM is also often associated with reduction in the level of packing of membrane phospholipids and an increase in membrane fluidity.
These alterations play an important role in making the cell surface a catalytic platform for the assembly of several clotting factor complexes, such as the tenase and prothrombinase protein complexes. Thus, PNOM occurring in platelets upon constitutes an important factor in normal blood coagulation, as well as in the initiation and/or propagation of abnormal, excessive blood clotting in numerous disorders. These disorders include, among others, arterial or venous thrombosis or thrombo-embolism.
Apoptosis is another major situation in which PNOM takes place. Apoptosis is an intrinsic program of cell self-destruction or “suicide”, which is inherent in every eukaryotic cell. In response to a triggering stimulus, cells undergo a highly characteristic cascade of events of cell shrinkage, blebbing of cell membranes, chromatin condensation and fragmentation, culminating in cell conversion to clusters of membrane-bound particles (apoptotic bodies), which are thereafter engulfed by macrophages. PNOM is a universal phenomenon in apoptosis, it occurs early in the apoptotic cascade, and has also been shown to be an important factor in the recognition and removal of apoptotic cells by macrophages.
Annexin V is a 37 kDa protein, capable of selective binding to apoptotic cells by binding to PS headgroups emerging on the surface of these cells as the result of the apoptosis-related PNOM process. However, annexin V is a relatively large protein, requiring relatively complex procedures of synthesis and marker attachment. Moreover, annexin V binds only to the surface of the cell in early apoptosis and can not perform accumulation within the cell at that stage.
It is therefore desirable to have small-molecular weight compounds, capable of selective targeting, binding and accumulation within cells undergoing PNOM (PNOM-cells). Such compounds can have applications in molecular imaging of apoptosis and blood clotting. Such compounds can also have useful therapeutic applications, when attached to therapeutically-useful drugs, by functioning as targeting moieties, enabling the active drug to target, bind and accumulate specifically in cells undergoing PNOM, thus allowing enhanced concentrations of the active drug in foci of disease.